Eosinophils play prominant roles in a variety of atopic conditions including allergic rhinitis, dermatitis, conjunctivitis, and particularly bronchial asthma (for a reviews see e.g. Gleich, G. J., et al., Eosinophils. J. I. Gallin, I. M. Goldstein, R. Snyderman, Eds., Inflammation: Basic Principles and Clinical Correlates (Raven Press, Ltd., New York, 1992) and Seminario, M. C., et al. (1994) Current Opinion in Immunology 6, 860-864). A pivotal event in the process is the accumulation of eosinophils at the involved sites. While a number of the classical chemoattractants, including C5a, LTB4, and PAF, are known to attract eosinophils (Gleich, G. J., et al., Eosinophils. J. I. Gallin, et al. Eds., Inflammation: Basic Principles and Clinical Correlates (Raven Press, Ltd., New York, 1992)), these mediators are promiscuous, acting on a variety of leukocytes including neutrophils, and are unlikely to be responsible for the selective accumulation of eosinophils. In contrast, the chemokines a family of 8-10 kDa proteins are more restricted in the leukocyte subtypes they target and are potential candidates for the recruitment of eosinophils in atopic diseases and asthma (Baggiolini, M., Dewald, B. and Moser, B. (1994) Advances in Immunology 55, 97-179). Although there is a mounting body of evidence that eosinophils are recruited to sites of allergic inflammation by a number of .beta.-chemokines, particularly eotaxin and RANTES, the receptor which mediates these actions has not been identified.
The chemokines contain four conserved cysteines, and are divided into two sub-families based on the arrangement of the first cysteine pair (Baggiolini, M., Dewald, B. and Moser, B. (1994) Advances in Immunology 55, 97-179). In the .alpha.-chemokine family, which includes IL-8, MGSA, NAP-2 and IP-10, these two cysteines are separated by a single amino acid, while in the .beta.-chemokine family, which includes RANTES ("regulated on activation T expressed and secreted"), MCP-1 ("monocyte chemotactic protein"), MCP-2, MCP-3, MIP-1.alpha. ("macrophage inflammatory protein"), MIP-1.beta. and eotaxin, these two cysteines are adjacent. There is a functional correlate to this structural division. The .alpha.-chemokines act primarily on neutrophils, and the .beta.-chemokines on monocytes, lymphocytes, basophils and eosinophils (Baggiolini, M., Dewald, B. and Moser, B. (1994) Advances in Immunology 55, 97-179). In particular, RANTES, MCP-2, MCP-3, and MIP-1.alpha. have been shown to activate eosinophils in vitro (Dahinden, C. A., et al. (1994) Journal of Experimental Medicine 179, 751-756; Ebisawa, M., et al. (1994) Journal of Immunology 153, 2153-2160; Weber, M., et al. (1995) Journal of Immunology 154, 4166-4172), and RANTES to selectively attract eosinophils in vivo (Meurer, R., et al. (1993) Journal of Experimental Medicine 178, 1913-1921; Beck, L., et al. (1995) FASEB Journal 9, A804). Similarly, eotaxin, a new member of the .beta.-chemokine family, first described in guinea pigs (Griffiths-Johnson, D. A., et al. (1993) Bichemical and Biophysical Research Communications 197, 1167-1172; Jose, P. J., et al. (1994) Journal of Experimental Medicine 179, 881-887) and mice (Rothenberg, et al. (1995) Proceedings of the National Academy of Sciences 92, 8960-8964) is also a potent attractant and activator of eosinophils both in vitro and in vivo. Moreover, eotaxin is generated during antigenic challenge in the guinea pig model of allergic airway inflammation (Jose, et al. (1994) J. Exp. Med., 179, 881-887; Rothenberg, et al. (1995) J. Exp. Med., 181, 1211-1216. The cloning of guinea pig eotaxin has been disclosed (PCT Patent Publication No. WO 95/07985; Mar. 23, 1995). The cloning of the human eosinophil chemoattractant eotaxin has recently been reported (Ponath, et al., J. Clin. Invest. (1996) 97(3) 604-612) and eotaxin has been suggested to be a very important agent in the mechanism of allergic inflammation (Baggiolini, et al., J. Clin. Invest. (1996) 97(3) 587).
Eosinophils are attracted by a number of .beta.-chemokines, the most potent of which are eotaxin (Griffiths-Johnson, D. A., et al. (1993) Bichemical and Biophysical Research Communications 197, 1167-1172; Jose, P. J., et al. (1994) Journal of Experimental Medicine 179, 881-887; Rothenberg, et al. (1995) Proceedings of the National Academy of Sciences 92, 8960-8964) and RANTES (Dahinden, C. A., et al. (1994) Journal of Experimental Medicine 179, 751-756; Ebisawa, M., et al. (1994) Journal of Immunology 153, 2153-2160; Weber, M., et al. (1995) Journal of Immunology 154, 4166-4172; Meurer, R., et al. (1993) Journal of Experimental Medicine 178, 1913-1921; Beck, L., et al. (1995) FASEB Journal 9, A804). Although several human .beta.-chemokine receptors have been characterized in detail, none have the appropriate selectivity to account for the observed responses.
While elucidation of the actions of .beta.-chemokines on eosinophils has contributed greatly to the understanding of eosinophil biology, information regarding the cell surface receptors which mediate these effects remain sparse. Furthermore, there are no reports describing binding studies of any of the .beta.-chemokines to primary eosinophils. The known .beta.-chemokine receptors are members of the G protein-coupled receptor superfarnily. Two of these receptors, CC CKR1 (12, 13) and CC CKR2 (MCP-1R) (Charo, I. F., et al. (1994) Proceecing of the National Academy of Sciences 91, 2752-2756; Myers, S. J., et al. (1995) Journal of Biological Chemistry 270, 5786-5792; Franci, C., et al. (1995) Journal of Immunology 154, 6511-6517) found on monocytes, have been extensively studied and their selectivity for the different chemokines defined. However, neither of these receptors has the necessary ligand selectivity or the appropriate expression patterns required to mediate the effects of the .beta.-chemokines on eosinophils. For example, CC CKR1 binds RANTES with high affinity, but binds eotaxin poorly, and while the effects of eotaxin on CC CKR2 have not been studied this receptor has no avidity for RANTES (Myers, S. J., et al. (1995) Journal of Biological Chemistry 270, 5786-5792).
A review of the role of chemokines in allergic inflammation is provided by Kita, H., et al., J. Exp. Med. 183, 2421-2426 (June 1996). In particular, this review discusses the role which the receptor CKR-3 plays in the process of allergic inflammation. The cloning, expression and characterization of the human eosinophil eotaxin receptor has been reported by Daugherty, B. J., et al., J. Exp. Med. 183, 2349-2354 (May 1996). This publication discloses the cloning and functional expression of the chemokine receptor CC CKR3, as well as its characterization.
The cloning and expression of a human eosinophil receptor was allegedly achieved by Combadiere, C., et al., J. Biological Chem. 270 (27), 16491-16494 (Jul. 14, 1995). However, in a subsequent retraction (J. Biological Chem. 270, 30235 (1995)) they confirmed that the receptor which was actually cloned and expressed was not CC CKR3, but was another CC chemokine receptor CC CKR5. This receptor was subsequently characterized by Kitaura, M., et al., J. Biological Chem. 271 (13), 7725-7730 (Mar. 29, 1996).
A human eotaxin receptor has been reported by Ponath, P. D., et al. J. Exp. Med. 183, 2437-2448 (June 1996) and Gerard, C. J., et al., PCT Publication No. WO 96/22371 (Jul. 25, 1996). However, the sequence disclosed in this publication possesses an error in the assignment of threonine rather than serine at position #276 of the receptor. In addition, functionality of the receptor was not fully demonstrated.
A retrovirus designated human immunodeficiency virus (HIV-1) is the etiological agent of the complex disease that includes progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system. This virus was previously known as LAV, HTLV-III, or ARV. Entry of HIV-1 into a target cell requires cell-surface CD4 and additional host cell cofactors. Fusin has been identified as a cofactor required for infection with virus adapted for growth in transformed T-cells, however, fusin does not promote entry of macrophagetropic viruses which are believed to be the key pathogenic strains of HIV in vivo. It has recently been recognized that for efficient entry into target cells, human immunodeficiency viruses require the chemokine receptors CCR-5 and CXCR-4, as well as the primary receptor CD4 (Levy, N. Engl. J. Med., 335(20), 1528-1530 (Nov. 14 1996). The principal cofactor for entry mediated by the envelope glycoproteins of primary macrophage-trophic strains of HIV-1 is CCR5, a receptor for the .beta.-chemokines RANTES, MIP-1.alpha. and MIP-1.beta. (Deng, et al., Nature, 381, 661-666 (1996)). HIV attaches to the CD4 molecule on cells through a region of its envelope protein, gp120. It is believed that the CD-4 binding site on the gp120 of HIV interacts with the CD4 molecule on the cell surface, and undergoes conformational changes which allow it to bind to another cell-surface receptor, such as CCR5 and/or CXCR-4. This brings the viral envelope closer to the cell surface and allows interaction between gp41 on the viral envelope and a fusion domain on the cell surface, fusion with the cell membrane, and entry of the viral core into the cell. It has been shown that .beta.-chemokine ligands prevent HIV-1 from fusing with the cell (Dragic, et al., Nature, 381, 667-673 (1996)). It has further been demonstrated that a complex of gp120 and soluble CD4 interacts specifically with CCR-5 and inhibits the binding of the natural CCR-5 ligands MIP-1.alpha. and MIP-1.beta. (Wu, et al., Nature, 384, 179-183 (1996); Trkola, et al., Nature, 384, 184-187 (1996)).
Humans who are homozygous for mutant CCR-5 receptors which do not serve as co-receptors for HIV-1 in vitro apper to be unusually resistant to HIV-1 infection and are not immunocompromised by the presence of this genetic variant (Nature, 382, 722-725 (1996)). Absence of CCR-5 appears to confer protection from HIV-1 infection (Nature, 382, 668-669 (1996)). Other chemokine receptors may be used by some strains of HIV-1 or may be favored by non-sexual routes of transmission. Although most HIV-1 isolates studied to date utilize CCR-5 or fusin, some can use both as well as the related CCR-2B and CCR-3 as co-receptors (Nature Medicine, 2(11), 1240-1243 (1996)). The determination that chemokine receptors are critical co-receptors for the entry of HIV into cells was pronounced a "1996 Breakthrough of the Year" by Science Magazine (Science, 274, 1987-1991 (Dec. 20, 1996)).
The use of orally-active agents which modulate the action of the eosinophil eotaxin receptor would be a significant advance in the treatment and prevention of atopic conditions including allergic rhinitis, dermatitis, conjunctivitis, and particularly bronchial asthma. Further, agents which could block the eosinophil eotaxin receptor in humans who possess normal chemokine receptors should prevent infection in healthy individuals and slow or halt viral progression in infected patients.
It would also be desirable to know the molecular structure of the eosinophil eotaxin receptor in order to analyze this new receptor family and understand its normal physiological role. This could lead to a better understanding of the in vivo processes which occur upon ligand-receptor binding. Further, it would be desirable to use cloned-eosinophil eotaxin receptor as essential components of an assay system which can identify new agents for the treatment and prevention of atopic conditions.